A sensor capable of super-sensitive and rapid detection of alpha-fetal protein and a preparation method and application thereof

Abstract

This invention discloses an ultrasensitive and rapid sensor for detecting alpha-fetoprotein (AFP), constructed based on a sandwich immunoassay structure and the electrochemiluminescence resonance energy transfer (ECL-RET) principle. The advantages of this invention compared to existing technologies are: high sensitivity and ultra-low detection limit: This invention innovatively uses MIL-NH2-RuMOF@Pt as an energy donor, whose MOF structure can load a large amount of Ru(bpy)3. 2+ The luminescent molecule provides a strong and stable initial ECL signal; ZnO@PDA acts as the acceptor, with its absorption spectrum highly overlapping with the donor emission spectrum, and PDA modification improves biocompatibility and coupling efficiency. The highly efficient RET pair formed by these two molecules, combined with the sandwich immunoamplification effect, achieves efficient signal quenching, resulting in a detection limit as low as 1.1 × 10⁻⁶. ‑ 14 mg·mL⁻¹; wide linear range; excellent specificity and accuracy; good stability and repeatability; broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of alpha-fetoprotein sensor technology, specifically to an ultrasensitive and rapid alpha-fetoprotein sensor, its preparation method, and its application. Background Technology

[0002] The Importance of AFP Testing: AFP is a key serum tumor marker widely used in clinical practice for important diseases such as primary liver cancer. Its concentration level is of great value for early screening, diagnosis, treatment evaluation, and prognostic monitoring of liver cancer. Therefore, developing a highly sensitive, highly specific, rapid, and easy-to-use AFP detection method is of urgent clinical need and significant social importance.

[0003] Current detection technologies and limitations: The mainstream detection methods for AFP currently include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CIA), and electrochemiluminescence immunoassay (ECIA). Among these, EIA combines the advantages of controllable electrochemistry with the absence of background light in chemiluminescence, resulting in high sensitivity. However, further improving detection sensitivity, lowering the detection limit, broadening the linear range, and enhancing sensor stability and anti-interference capabilities remain goals for this field.

[0004] ECL-RET technology: Electrochemiluminescence resonance energy transfer is a novel signal transduction and amplification strategy. Its principle is that when the ECL emission spectrum of the energy donor and the absorption spectrum of the energy acceptor have sufficient overlap and are sufficiently close (typically <10 nm), the excited-state energy of the donor will be non-radiatively transferred to the acceptor, resulting in the quenching (or enhancement) of the donor's ECL signal. Constructing "signal switch" or "signal amplification" type sensors based on ECL-RET is an effective way to improve detection performance.

[0005] Current technological limitations: In ECL-RET-based sensors, finding efficient and stable energy donor-acceptor pairs and achieving stable and efficient coupling with biorecognition elements (such as antibodies), while ensuring the efficient execution of the RET process, remains a technical challenge. Some existing donor-acceptor pairs may suffer from poor spectral overlap, low energy transfer efficiency, poor biocompatibility, or complex modification processes. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a novel, highly efficient electrochemiluminescence biosensor for detecting AFP based on the ECL-RET energy pair. This sensor exhibits extremely high sensitivity, excellent specificity, good stability, and a wide linear range, making it suitable for the accurate detection of AFP in complex serum samples.

[0007] To solve the above-mentioned technical problems, the technical solution provided by this invention is: a sensor capable of ultrasensitive and rapid detection of alpha-fetoprotein, constructed based on a sandwich immune structure and the principle of electrochemiluminescence resonance energy transfer (ECL-RET), comprising: Working electrode; The energy donor-antibody complex (MIL-NH2-RuMOF@Pt-Ab1) immobilized on the surface of the working electrode is a tris(2,2′-bipyridine)ruthenium(II) (Ru(bpy)3) 2+ The composite material is an amino-metal-organic framework-platinum composite, wherein the composite material is immobilized with alpha-fetoprotein primary antibody by covalent coupling, and the ECL emission spectrum of MIL-NH2-RuMOF@Pt is in the range of 550-680 nm. Energy receptor-antibody complex (ZnO@PDA-Ab2): The energy receptor is a dopamine-modified zinc oxide nanomaterial (ZnO@PDA), whose UV-Vis absorption spectrum covers the 500-700 nm band and significantly overlaps with the emission spectrum of MIL-NH2-RuMOF@Pt. The complex is covalently coupled with alpha-fetoprotein secondary antibody (Ab2).

[0008] Furthermore, the working electrode is a glassy carbon electrode.

[0009] Furthermore, the covalent coupling method is a glutaraldehyde crosslinking reaction or a carboxyl-amino condensation reaction.

[0010] Furthermore, the sensor has a linear detection range of 1.0 × 10⁻⁶ for alpha-fetoprotein. -13 mg·mL -1 Up to 1.0×10 -5 mg·mL -1 The limit of detection is 1.1 × 10⁻⁶. -14 mg·mL -1 (Signal-to-noise ratio S / N=3).

[0011] A method for preparing an ultrasensitive and rapid alpha-fetoprotein sensor includes the following steps: Step 1: Preparation of the MIL-NH2-RuMOF@Pt-Ab1 complex; 1) MIL-NH2MOF was synthesized by mixing ruthenium bipyridine, aluminum chloride hexahydrate and 2-aminoterephthalic acid in a molar ratio of 4:3:4, dissolving them in N,N-dimethylformamide, and reacting them in a reactor at 130°C for 72 hours. Platinum nanoparticles (PtNPs) were prepared by reacting 20 mmol chloroplatinic acid with 10 mmol ascorbic acid at 4 °C for 2 hours. They were then uniformly dispersed on the surface of MIL-NH2MOF by stirring to obtain MIL-NH2MOF@Pt. Ru(bpy)3 can be encapsulated in situ or modified by post-synthesis. 2+ Loading it into MIL-NH2MOF@Pt yields MIL-NH2-RuMOF@Pt; 4) Using the amino groups on the surface of MIL-NH2-RuMOF@Pt, the alpha-fetoprotein primary antibody (Ab1) was immobilized by covalent coupling reaction to obtain the MIL-NH2-RuMOF@Pt-Ab1 complex. Step 2: Preparation of ZnO@PDA-Ab2 complex; 1) Prepare 0.5 mol·L⁻¹ -1 A Zn(NO3)2 aqueous solution was added dropwise with 10 mol·L⁻¹ -1 The solution was prepared with NaOH solution until pH=13, stirred until clear, and then dried to obtain ZnO powder. 2) Prepare a Tris-HCl buffer solution with pH=8.5, dissolve 10 mg of dopamine in it, and add 1 mL of 1 mg / mL solution. -1 ZnO solution was stirred at room temperature for more than 30 minutes to obtain ZnO@PDA via dopamine self-polymerization reaction; 3) Disperse ZnO@PDA in PBS solution at pH=7.4, add 0.25% glutaraldehyde and react at 37℃ for 30 minutes to activate it, bind it with alpha-fetoprotein secondary antibody (Ab2) at room temperature and shake overnight, centrifuge and wash, and block non-specific binding sites with 1% bovine serum albumin to obtain ZnO@PDA-Ab2 complex. Step 3: Modify the working electrode; 1) Polish and clean the working electrode as a pretreatment. 2) The MIL-NH2-RuMOF@Pt-Ab1 complex was drop-coated onto the electrode surface and dried and fixed; 3) 1% bovine serum albumin solution was added to block non-specific binding sites, and the sensor was obtained after rinsing with PBS solution.

[0012] Furthermore, the covalent coupling reaction described in step 1 is a glutaraldehyde crosslinking reaction or a carboxyl-amino condensation reaction.

[0013] Furthermore, in step 2, the binding of ZnO@PDA with Ab2 is achieved through a Schiff base reaction.

[0014] Furthermore, the reaction conditions for blocking non-specific binding sites in step 3 are incubation at 37°C for 30 minutes.

[0015] An application of an ultrasensitive and rapid alpha-fetoprotein (AFP) sensor in detecting AFP in human serum, and another application of an ultrasensitive and rapid alpha-fetoprotein (AFP) sensor in detecting other disease-related protein biomarkers, nucleic acids, or small molecules, wherein the primary antibody (Ab1) and secondary antibody (Ab2) of AFP in the sensor are replaced with specific antibodies corresponding to the target analytes.

[0016] With the above structure, the present invention has the following advantages: high sensitivity and ultra-low detection limit: The present invention innovatively uses MIL-NH2-RuMOF@Pt as an energy donor, and its MOF structure can load a large amount of Ru(bpy)3. 2+ The luminescent molecule provides a strong and stable initial ECL signal; ZnO@PDA acts as the acceptor, with its absorption spectrum highly overlapping with the donor emission spectrum, and PDA modification improves biocompatibility and coupling efficiency. The highly efficient RET pair formed by these two molecules, combined with the sandwich immunoamplification effect, achieves efficient signal quenching, resulting in a detection limit as low as 1.1 × 10⁻⁶. -14 mg·mL⁻¹.

[0017] Wide linear range: The sensor's response to the AFP is within 1.0 × 10⁻⁶. -13 mg·mL -1 Up to 1.0×10 -5 mg·mL -1 It exhibits a good linear relationship within the concentration range, spanning eight orders of magnitude, and can meet the detection needs of clinical samples from trace amounts to high concentrations of AFP.

[0018] Excellent specificity and accuracy: Experiments show that the sensor has no significant response to common interfering substances such as carcinoembryonic antigen, prostate-specific antigen, and human serum albumin, demonstrating high specificity for AFP recognition. In human serum spiked recovery experiments, the recovery rate ranged from 98.1% to 113.7%, indicating its accuracy and reliability in detecting complex matrices.

[0019] Good stability and repeatability: The MIL-MOF framework and ZnO@PDA material exhibit good chemical stability, and the ECL signal of the modified electrode remains stable after multiple cyclic scans and long-term storage. Sensors prepared in different batches show small relative standard deviations in response to the same concentration of AFP, demonstrating good repeatability.

[0020] Broad application prospects: The core of this invention lies in the construction of a highly efficient ECL-RET immunosensing platform. By replacing the conjugated antibody, this platform is expected to be used for the sensitive detection of other disease-related protein biomarkers, nucleic acids, small molecules, etc., and has broad versatility and promotional value. Attached Figure Description

[0021] Figure 1This is a schematic diagram of the structure of the sensor of the present invention.

[0022] Figure 2 The following are schematic diagrams of (A) MIL MOF, MIL RuMOF@Pt XRD; (B) MIL MOF XRD; (C) ZnO@PDA XRD; and (D) ZnO@PDA XRD.

[0023] Figure 3 This diagram shows the ECL of the sensor for different concentrations of AFP, as well as the electrochemiluminescence meter, electrochemical impedance spectroscopy, CV curves, and a schematic diagram of condition optimization. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the accompanying drawings.

[0025] Combined with appendix Figure 1-3 An electrochemiluminescence biosensor for detecting alpha-fetoprotein (AFP) is constructed based on a sandwich immunostructure and the electrochemiluminescence resonance energy transfer principle, comprising: Working electrode: Usually a solid electrode such as a glassy carbon electrode.

[0026] Energy donor-antibody complex (Ab1-donor) immobilized on the working electrode: The energy donor is an amino-based metal-organic framework material loaded with tris(2,2′-bipyridine)ruthenium(II), denoted as MIL-NH2-RuMOF@Pt. This complex, denoted as MIL-NH2-RuMOF@Pt-Ab1, is immobilized with an alpha-fetoprotein primary antibody via covalent coupling or other methods. MIL-NH2-RuMOF@Pt exhibits strong and stable ECL emission, with an emission spectrum ranging from 550 to 680 nm.

[0027] Energy receptor-antibody complex (Ab2-receptor): The energy receptor is a dopamine-modified zinc oxide nanomaterial, denoted as ZnO@PDA. The UV-Vis absorption spectrum of ZnO@PDA covers the 500-700 nm band and significantly overlaps with the emission spectrum of MIL-NH2-RuMOF@Pt, satisfying the conditions for ECL-RET. This receptor is labeled with an alpha-fetoprotein secondary antibody via covalent coupling, denoted as ZnO@PDA-Ab2.

[0028] V. Detection Principle: In the absence of the target analyte AFP, the energy acceptor ZnO@PDA-Ab2 is far from the energy donor MIL-NH2-RuMOF@Pt-Ab1 on the electrode surface, RET does not occur, and the sensor generates a strong initial ECL signal.

[0029] When the target analyte AFP is present, the AFP antigen binds simultaneously to Ab1 on the electrode and Ab2 in the solution, forming a sandwich immune complex of "working electrode / MIL-NH2-RuMOF@Pt-Ab1 / AFP / ZnO@PDA-Ab2". This brings the energy receptor ZnO@PDA closer to the energy donor MIL-NH2-RuMOF@Pt, resulting in highly efficient ECL-RET within an effective distance (typically within 10 nm). The donor's energy is absorbed by the receptor, causing significant quenching of the ECL signal on the electrode surface. Quantitative analysis of AFP can be achieved by measuring the degree of ECL signal quenching (ΔECL).

[0030] The present invention also provides a method for manufacturing the sensor, comprising the following steps: (1) Preparation of MIL-NH2-RuMOF@Pt-Ab1 complex: First, MIL-NH2MOF@Pt was synthesized, and Ru(bpy)3 was encapsulated in situ or modified by post-synthesis. 2+ The MIL-NH2-RuMOF@Pt was loaded into the substrate to obtain MIL-NH2-RuMOF@Pt. Then, using the amino groups on its surface, alpha-fetoprotein primary antibody (Ab1) was covalently coupled through reactions such as glutaraldehyde crosslinking or carboxyl-amino condensation.

[0031] (2) Preparation of ZnO@PDA-Ab2 complex: First, ZnO nanomaterials (such as nanospheres and nanorods) are synthesized, and a polydopamine layer is coated on their surface by self-polymerization to obtain ZnO@PDA. Utilizing the abundant active groups of PDA (such as amino and phenolic hydroxyl groups), alpha-fetoprotein secondary antibody (Ab2) is covalently coupled through a similar method.

[0032] (3) Modification of the sensor working electrode: The glassy carbon electrode was pretreated by polishing and cleaning. The MIL-NH2-RuMOF-Ab1 complex was drop-coated onto the electrode surface and dried and fixed. Then, non-specific binding sites were blocked with bovine serum albumin solution to obtain a sensing interface that can be used for detection.

[0033] This invention further provides the application of the sensor in the detection of alpha-fetoprotein, particularly in the detection of AFP in complex biological samples such as human serum.

[0034] The experiment consisted of three main steps: synthesis of MIL RuMOF, preparation of platinum nanoparticles (PtNPs), and construction of MIL RuMOF@Pt composite materials. The specific procedures are as follows: Synthesis of MIL RuMOF: Ruthenium bipyridine, aluminum chloride hexahydrate, and 2-aminoterephthalic acid were mixed in a molar ratio of 4:3:4 and dissolved in N,N-dimethylformamide (DMF). The mixture was then transferred to a high-temperature reactor and reacted at 130°C for 72 hours to obtain the MIL RuMOF material.

[0035] Preparation of platinum nanoparticles: 20 mmol of chloroplatinic acid (H2PtC) was added. l6 Platinum nanoparticles were prepared by reacting 10 mmol of ascorbic acid (AA) with 10 mmol at 4°C for 2 hours via a reduction reaction.

[0036] (3) Construction of MIL RuMOF@Pt composite material: MIL RuMOF and PtNPs were mixed by stirring to make PtNPs uniformly dispersed on the surface of MIL RuMOF, and finally MIL RuMOF@Pt composite material was obtained.

[0037] Preparation of ZnO powder: First, prepare 0.5 mol·L⁻¹ ZnO powder. -1 A Zn(NO3)2 aqueous solution was prepared, and then 10 mol·L⁻¹ was added dropwise to the solution. -1 A large amount of flocculent precipitate was rapidly formed in the beaker when the NaOH solution was added. The solution was stirred continuously until it became clear, and the pH was finally adjusted to 13. The resulting solution was dried to obtain ZnO powder. Preparation of ZnO@PDA composite material: First, a Tris-HCl buffer solution with pH=8.5 was prepared. 10 mg of dopamine was accurately weighed and dissolved in the above Tris-HCl buffer solution. Simultaneously, a 1 mg·mL⁻¹ solution was prepared… -1 One mL of a ZnO solution was added to a dopamine Tris-HCl solution. The mixture was stirred continuously at room temperature for at least 30 minutes. Through the self-polymerization reaction of dopamine, the ZnO@PDA composite material was finally obtained.

[0038] The preparation of the Ab2-ZnO@PDA complex mainly includes the following steps: 2 mg ZnO@PDA was dispersed in 1 mL of PBS solution (pH=7.4), 0.25% glutaraldehyde (GA) was added, and the mixture was reacted at 37°C for 30 minutes to complete activation. The cross-linking agent was then removed, and the mixture was shaken overnight at room temperature. The activated ZnO@PDA was then reacted with 1 mL of 1.0×10⁻⁶ PBS solution. -6 mg·mL -1Ab2 was mixed with ZnO@PDA, and the Schiff base reaction on the ZnO@PDA surface was used to bind Ab2 to it. The complex was collected by centrifugation and washed with PBS (pH=7.4) to remove unbound Ab2. The final product was stored at 4°C for later use. To reduce non-specific binding of Ab2-ZnO@PDA to other proteins, 100 µL of 1% bovine serum albumin was mixed with the complex and shaken at room temperature for 30 minutes. Subsequently, it was washed with PBS solution to remove unreacted BSA, and the final product was dispersed in 1 mL of PBS solution (pH=7.4) for later use. Through the above steps, the Ab2-ZnO@PDA complex was successfully constructed, laying the foundation for subsequent applications.

[0039] This study constructed a ZnO@PDA-mediated RET-ECL system through the following steps: The sensor construction process includes the following steps: Non-specific site blocking: On a cleaned glassy carbon electrode, first use 20 μL of 0.25% GA and 20 μL of 1 mg·mL⁻¹. -1 The amide reaction was carried out using MIL NH2-RuMOF@Pt, with 20 μL of 1.0×10⁻⁶ solution added dropwise. -8 mg·mL -1 Primary antibody (Ab1) was used to form a MIL NH2-RuMOF@Pt-Ab1 bioconjugate. MIL NH2-RuMOF@Pt-Ab1 was modified onto a glassy carbon electrode. 10 μL of 1% BSA was added to the MILNH2-RuMOF@Pt-Ab1 modified electrode and the electrode was incubated at 37°C for 30 minutes to block non-specific binding sites. Excess BSA was removed with PBS.

[0040] AFP binding: 20 μL of different concentrations (1.0 × 10⁻⁶) were added to the electrode. 13 -1.0×10 -5 mg·mL -1 AFP was incubated at 4°C for 12 hours to ensure specific binding of AFP to Ab1, and then unreacted AFP was removed with PBS.

[0041] (3) Ab2-ZnO@PDA binding: 20 μL of 1.0×10 -6 mg·mL -1The Ab2-ZnO@PDA bioconjugate was dropped onto the electrode and incubated at 37°C for 30 minutes to allow Ab2-RuMOF to bind to AFP. Finally, the electrode was thoroughly rinsed with PBS solution at pH 7.4. After completing these steps, the ZnO@PDA-mediated RET-ECL system was constructed and stored at 4°C for further use. This method successfully established a highly efficient RET-ECL system through optimized electrode treatment, blocking of non-specific sites, and specific binding to AFP.

[0042] This invention successfully combines functionalized MIL NH2-RuMOF@Pt with the luminescence quencher ZnO@PDA, proposing a novel ECL signal quenching strategy. This not only provides an efficient tool for the high-sensitivity detection of AFP, but also offers new material design and technical ideas for developing a multifunctional ECL sensing platform targeting other disease biomarkers.

[0043] The present invention and its embodiments have been described above. This description is not restrictive, and the actual structure is not limited thereto. In conclusion, if those skilled in the art, inspired by this description, design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the scope of protection of this invention.

Claims

1. A sensor capable of ultrasensitive and rapid detection of alpha-fetoprotein, characterized in that: Constructed based on the sandwich immune structure and the principle of electrochemiluminescence resonance energy transfer (ECL-RET), including: Working electrode; The energy donor-antibody complex (MIL-NH2-RuMOF@Pt-Ab1) immobilized on the surface of the working electrode is a tris(2,2′-bipyridine)ruthenium(II) (Ru(bpy)3) 2+ The composite material is an amino-metal-organic framework-platinum composite, wherein the composite material is immobilized with alpha-fetoprotein primary antibody by covalent coupling, and the ECL emission spectrum of MIL-NH2-RuMOF@Pt is in the range of 550-680 nm. Energy receptor-antibody complex (ZnO@PDA-Ab2): The energy receptor is a dopamine-modified zinc oxide nanomaterial (ZnO@PDA), whose UV-Vis absorption spectrum covers the 500-700 nm band and significantly overlaps with the emission spectrum of MIL-NH2-RuMOF@Pt. The complex is covalently coupled with alpha-fetoprotein secondary antibody (Ab2).

2. The sensor for ultrasensitive and rapid detection of alpha-fetoprotein according to claim 1, characterized in that: The working electrode is a glassy carbon electrode.

3. The sensor for ultrasensitive and rapid detection of alpha-fetoprotein according to claim 1, characterized in that: The covalent coupling method is either a glutaraldehyde crosslinking reaction or a carboxyl-amino condensation reaction.

4. The sensor for ultrasensitive and rapid detection of alpha-fetoprotein according to claim 1, characterized in that: The sensor has a linear detection range of 1.0 × 10⁻⁶ for alpha-fetoprotein. -13 mg·mL -1 Up to 1.0×10 -5 mg·mL -1 The limit of detection is 1.1 × 10⁻⁶. -14 mg·mL -1 (Signal-to-noise ratio S / N=3).

5. A method for manufacturing a sensor as described in any one of claims 1-4, characterized in that: Includes the following steps: Step 1: Preparation of the MIL-NH2-RuMOF@Pt-Ab1 complex; 1) MIL-NH2MOF was synthesized by mixing ruthenium bipyridine, aluminum chloride hexahydrate and 2-aminoterephthalic acid in a molar ratio of 4:3:4, dissolving them in N,N-dimethylformamide, and reacting them in a reactor at 130°C for 72 hours. 2) Platinum nanoparticles (PtNPs) were prepared by reacting 20 mmol chloroplatinic acid with 10 mmol ascorbic acid at 4 °C for 2 hours. They were then uniformly dispersed on the surface of MIL-NH2MOF by stirring to obtain MIL-NH2MOF@Pt. 3) Use in-situ encapsulation or post-synthesis modification methods to modify Ru(bpy)3 2+ Loading it into MIL-NH2MOF@Pt yields MIL-NH2-RuMOF@Pt; 4) Using the amino groups on the surface of MIL-NH2-RuMOF@Pt, the alpha-fetoprotein primary antibody (Ab1) was immobilized by covalent coupling reaction to obtain the MIL-NH2-RuMOF@Pt-Ab1 complex. Step 2: Preparation of ZnO@PDA-Ab2 complex; 1) Prepare 0.5 mol·L⁻¹ -1 A Zn(NO3)2 aqueous solution was added dropwise with 10 mol·L⁻¹ -1 The solution was prepared with NaOH solution until pH=13, stirred until clear, and then dried to obtain ZnO powder. 2) Prepare a Tris-HCl buffer solution with pH=8.5, dissolve 10 mg of dopamine in it, and add 1 mL of 1 mg / mL solution. -1 ZnO solution was stirred at room temperature for more than 30 minutes to obtain ZnO@PDA via dopamine self-polymerization reaction; 3) Disperse ZnO@PDA in PBS solution at pH=7.4, add 0.25% glutaraldehyde and react at 37℃ for 30 minutes to activate it, bind it with alpha-fetoprotein secondary antibody (Ab2) at room temperature and shake overnight, centrifuge and wash, and block non-specific binding sites with 1% bovine serum albumin to obtain ZnO@PDA-Ab2 complex. Step 3: Modify the working electrode; 1) Polish and clean the working electrode as a pretreatment. 2) The MIL-NH2-RuMOF@Pt-Ab1 complex was drop-coated onto the electrode surface and dried and fixed; 3) 1% bovine serum albumin solution was added to block non-specific binding sites, and the sensor was obtained after rinsing with PBS solution.

6. The preparation method according to claim 5, characterized in that, The covalent coupling reaction described in step 1 is either a glutaraldehyde crosslinking reaction or a carboxyl-amino condensation reaction.

7. The preparation method according to claim 5, characterized in that, In step 2, the binding of ZnO@PDA with Ab2 is achieved through a Schiff base reaction.

8. The preparation method according to claim 5, characterized in that, The reaction conditions for blocking non-specific binding sites in step 3 are incubation at 37°C for 30 minutes.

9. The application of the sensor as described in any one of claims 1-4 in detecting alpha-fetoprotein in human serum.

10. An application of the sensor as described in any one of claims 1-4 in detecting other disease-related protein biomarkers, nucleic acids, or small molecules, characterized in that, Replace the alpha-fetoprotein primary antibody (Ab1) and secondary antibody (Ab2) in the sensor with specific antibodies corresponding to the target analyte.

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